Lung cancer is the leading cause of cancer-related death among both men and women in the United States and developed countries. More than 163,000 deaths due to lung cancer in the U.S. were estimated in 2007. With an overall 5-year survival rate of less than 15%, novel diagnostic and therapeutic strategies are critically needed; however, limitations in our understanding of the biology of the disease have hampered these efforts. Epigenetic alterations, particularly aberrant DNA methylation, play as important a role as genetic alterations in lung tumorigenesis. Disregulation of DNA methyltransferases, particularly DNMT1 and DNMT3B, has been implicated in the abnormal distribution of DNA methylation patterns in tumorigenesis. We have identified a novel DNMT3B subfamily, DNMT3B, with at least 7 transcriptional variants. We also discovered that a significant portion of lung cancers expressed only DNMT3B lacking the methyltransferase enzymatic domain (DNMT3B-del), suggesting an expanded role of DNMT3B in lung tumorigenesis. We further showed that DNMT3B is the major form of DNMT3B transcription in adult tissues, and that expression patterns of DNMT3B variants are critical for promoter-specific regulation of DNA methylation. Therefore, we hypothesize that DNMT3B plays an essential role in maintaining DNA methylation pattern distributions in the human genome, and that disrupted expression patterns of the DNMT3Bs determine abnormal DNA methylation with promoter-specificity in lung tumorigenesis. We propose 4 specific aims: Aim 1: To correlate expression patterns of DNMT3B and DNMT3B variants to the global and promoter-specific DNA methylation patterns in primary lung cancer specimens; Aim 2: To determine functional differences between DNMT3B and DNMT3B in DNA methylation control and other biological properties; Aim 3: To determine biological properties of the major DNMT3B variants with or without the DNA methyltransferase enzymatic domains; and Aim 4: To determine the causal relationship between DNMT3B variants and DNA methylation patterns in lung tumorigenesis. Our long-term goals are to better understand the role of these aberrant DNA methyltransferases and to apply this knowledge in future novel diagnostic and therapeutic strategies targeting DNA methylation in lung cancer.